title: Applicability of the product isolation and the radiometric aromatase assays for the measurement of low levels of aromatase: lack of aromatase activity in the human endometrium.
abstract: The purpose of this investigation was to assess the applicability of two well established procedures: (i) the product isolation assay and (ii) the radiometric 3H2O assay for the determination of very low levels of aromatase activity. The methods were validated and used to assess the capacity of normal and neoplastic human endometrium to synthesize oestrogens from androgens. Using the product isolation assay, various specimens (n = 27) of normal and neoplastic endometrium were incubated with [1,2,6,7-3H]testosterone either by a standard incubation procedure or by a superfusion technique. Following the incubation, carrier oestrone and oestradiol or [14C]oestrone and [14C]oestradiol were added, and the oestrogens were isolated and purified by paper chromatography and high-performance liquid chromatography. The radiochemical purity of oestrone and oestradiol was checked by the isotope dilution technique. In all samples, the 3H associated with oestrone and oestradiol failed to recrystallize as oestrone and oestradiol. No radioactivity was detectable in the oestrone and oestradiol crystals after acetylation. Similarly, 16 endometrial samples were tested for aromatase activity by the 3H2O release assay using [1 beta-3H]androstenedione as substrate. The results indicate that 3H2O was indeed released during these incubations, but this activity could not be inhibited by the aromatase inhibitor 4-hydroxyandrostenedione, by excess substrate or by heat inactivation of the tissue. Furthermore, the release of 3H2O from [1 beta-3H]androstenedione under the incubation conditions used (Dulbecco's modified Eagle's medium or RPMI-1640 containing fetal bovine serum and NADPH) also occurred in the absence of any tissue. This activity was not inhibited by 4-hydroxyandrostenedione nor by excess substrate. The results demonstrate that the human endometrium does not contain detectable levels of aromatase activity and that the radiometric assay can give rise to false-positive results if used for detection of very low levels of aromatase activity.
authors.affiliation: Department of Obstetrics and Gynaecology, St Michael's Hospital, University of Toronto, Ontario, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Prefontaine
authors.forename: C
authors.initials: C
authors.lastname: Shih
authors.forename: C C
authors.initials: CC
authors.lastname: Pan
authors.forename: B R
authors.initials: BR
authors.lastname: Bhavnani
mesh_terms: D000735:Androstenedione / Q000378:metabolism; D001141:Aromatase / Q000032:analysis* / Q000378:metabolism; D003470:Culture Media; D004717:Endometrium / Q000201:enzymology*; D005260:Female; D006801:Humans; D008722:Methods; D010920:Placenta / Q000201:enzymology; D011874:Radiometry / Q000379:methods; D014867:Water / Q000378:metabolism
chemical_list: D003470:Culture Media; D014867:Water; D000735:Androstenedione; D001141:Aromatase